Application of Rv1039c protein in preparation of antigen presentation inhibitor
By using Rv1039c protein to inhibit the expression of antigen-presenting cells surface molecules, the problem of antigen-presenting cells activation after Mycobacterial infection was solved, effective inhibition of adaptive immune responses was achieved, and the generation of immune rejection responses was avoided.
Patent Information
- Application Number
- CN202510390126.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art is difficult to effectively inhibit the activation of antigen presenting cells after Mycobacterium infection, resulting in the occurrence of an adaptive immune response.
By using the Rv1039c protein, the expression of antigen-presenting cell surface molecules, including transcription and protein expression of molecules such as MHC II, CD40, CD80 and CIITA, thereby inhibiting the occurrence of T cell activation and adaptive immune response.
The Rv1039c protein can significantly inhibit the secretion of IL-2, downregulate the expression of surface molecules of dendritic cells and macrophages, effectively inhibit the occurrence of adaptive immune responses, and do not produce excessive immune rejection reactions.
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Figure CN120204357A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to the application of Rv1039c protein in the preparation of antigen presentation inhibitors. Background Art
[0002] Mycobacterium tuberculosis (M. tuberculosis, M. tb) is the pathogen of tuberculosis and is mainly transmitted through respiratory aerosols. When M. tb breaks through the airway mucosal barrier and invades the lungs, it can trigger a local innate immune response, attracting macrophages, dendritic cells (DCs), neutrophils, etc. to aggregate at the infection site, forming a pathological structure characterized by granulomas. During this process, the maturation and functional activation of host antigen-presenting cells (APCs) are the key links in initiating adaptive immunity.
[0003] After mycobacteria infect the body, the immune response mediated by surface molecules of antigen-presenting cells is the core of clearing pathogens. Among them, molecules such as MHC class II molecules, CIITA, CD40, CD80 / CD86 on the surface of APCs play key roles in antigen presentation and T cell activation. During infection, pathogen-associated molecular patterns (PAMPs) activate APCs through Toll-like receptors (TLRs), induce the expression of CIITA, thereby upregulating MHC class II molecules and enhancing the antigen presentation ability of APCs. Infection signals synchronously induce APCs to express co-stimulatory molecules such as CD40, CD80 / CD86, providing a second signal for T cell activation. After the pathogen is taken up by APCs, it is processed into antigen peptides by lysosomes, combined with MHC class II molecules and presented on the surface of T cells. CD4+ T cells recognize the MHC II-antigen peptide complex through TCR, and at the same time receive co-stimulatory signals, differentiating into effector cells (such as Th1, Th2) or regulatory T cells.
[0004] Antigen presentation is an important link in activating the acquired immune response of the body after M. tb infection. M. tb has the ability to interfere with antigen presentation to prevent or alter T cell immune responses, thereby establishing latent or progressive infections. Therefore, deepening the understanding of how M. tb interferes with antigen presentation and subverts or escapes the acquired immune mechanism is crucial for the development of new host-directed therapies, disease biomarkers, and effective tuberculosis vaccines.
[0005] In summary, there is an urgent need in the art to develop an immunological active substance that can effectively inhibit antigen presentation after mycobacterial infection. Summary of the Invention
[0006] Objective of the Invention: The technical problem to be solved by the present invention is to provide the application of Rv1039c protein in the preparation of an antigen presentation inhibitor. This protein can inhibit the expression of surface molecules on antigen-presenting cells and has the function of inhibiting the occurrence of adaptive immune responses.
[0007] Technical Solution: The present invention provides the application of Rv1039c protein in the preparation of an antigen presentation inhibitor, and the amino acid sequence of the Rv1039c protein is shown as SEQ ID NO:1.
[0008] SEQ ID NO:1: MDFGALPPEINSARMYAGAGAGPMMAAGAAWNGLAAELGTT AASYESVITRLTTESWMGPASMAMVAAAQPYLAWLTYTAEAAAHAGSQAMASAAAYEAAYAMTVPPEVVAANRALLAALVATNVLGINTPAIMATEALYAEMWAQDALAMYGYAAASGAAGMLQPLSPPSQTTNPGGLAAQSAAVGSAAATAAVNQVSVADLISSLPNAVSGLASPVTSVLDSTGLSGIIADIDALLATPFVANIINSAVNTAAWYVNAAIPTAIFLANALNSGAPVAIAEGAIEAAEGAASAAAAGLADSVTPAGLGASLGEATLVGRLSVPAAWSTAAPATTAGATALEGSGWTVAAEEAGPVTGMMPGMASAAKGTGAYAGPRYGFKPTVMPKQVVV.
[0009] The present invention also provides the application of the gene encoding the above-mentioned Rv1039c protein in the preparation of an antigen presentation inhibitor.
[0010] Furthermore, the nucleotide sequence of the gene is shown as SEQ ID NO:2.
[0011]
[0012] The present invention also provides the use of a recombinant vector, recombinant bacterium or recombinant cell containing a gene encoding the Rv1039c protein in the preparation of an antigen presentation inhibitor, wherein the amino acid sequence of the Rv1039c protein is as shown in SEQ ID NO:1.
[0013] Furthermore, the nucleotide sequence of the gene is as shown in SEQ ID NO:2.
[0014] Furthermore, inhibiting antigen presentation includes inhibiting the secretion of T cell activation cytokines, and down-regulating the transcriptional level and protein expression level of surface molecules on dendritic cells and macrophages.
[0015] Furthermore, the T cell activation cytokine is IL-2.
[0016] Furthermore, the surface molecules are CD40, CD80, MHC II and CIITA.
[0017] Among them, the expression vector contains the aforementioned polynucleotide.
[0018] Methods well-known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA technology, DNA synthesis technology, etc. The DNA encoding the protein can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis and thus express the protein.
[0019] Preferably, the expression vector is a prokaryotic expression vector and a mycobacterial expression vector. Further, the prokaryotic expression vector can be stably expressed in Escherichia coli, such as the prokaryotic vector pET28a, etc. The mycobacterial expression vector can be stably expressed in Mycobacterium smegmatis, such as the mycobacterial vector pMV261.
[0020] Among them, the host cell contains or integrates the above expression vector.
[0021] The host cell can be a prokaryotic cell, such as a bacterial cell. Representative examples include: Escherichia coli, Mycobacterium smegmatis, etc.
[0022] The present invention also provides the use of a recombinant vector, recombinant bacterium or recombinant cell containing a gene encoding the Rv1039c protein in the preparation of a drug for treating mycobacterial infection, wherein the amino acid sequence of the Rv1039c protein is as shown in SEQ ID NO:1.
[0023] Furthermore, the nucleotide sequence of the gene is as shown in SEQ ID NO:2.
[0024] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The protein (Rv1039c) of the present invention will not cause excessive immune rejection reactions; secondly, Rv1039c can inhibit the expression of surface molecules of antigen-presenting cells and the occurrence of adaptive immune responses. Rv1039c targets not a single antigen presentation process, but multiple factors and multiple pathways, and potently inhibits the occurrence of adaptive immunity. Description of the Drawings
[0025] Figure 1 Showing that the Rv1039c protein inhibits the secretion of the T cell activation cytokine IL-2;
[0026] Figure 2 Showing that overexpression of Rv1039c inhibits the secretion of the T cell activation cytokine IL-2 induced by Mycobacterium smegmatis;
[0027] Figure 3 Showing that overexpression of Rv1039c inhibits the transcription of the dendritic cell surface molecules CD40, CD80, MHC II, and CIITA induced by Mycobacterium smegmatis.
[0028] Figure 4 Showing that overexpression of Rv1039c inhibits the transcription of the macrophage surface molecules CD40, CD80, MHCII, and CIITA induced by Mycobacterium smegmatis.
[0029] Figure 5 Showing that overexpression of Rv1039c downregulates the secretion of the dendritic cell surface molecule MHC II induced by Mycobacterium smegmatis.
[0030] Figure 6 Showing that overexpression of Rv1039c downregulates the secretion of the RAW264.7 cell surface molecule MHC II induced by Mycobacterium smegmatis. Detailed Embodiments
[0031] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings.
[0032] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques have been well described in the existing literature. For details, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.
[0033] Example 1 Secretion level of the T cell activation cytokine IL-2 inhibited by the Rv1039c protein
[0034] (1) Prokaryotic expression and identification of the Rv1039c protein
[0035] First, primers with BamH I and Hind III double restriction sites were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.:
[0036] Forward primer: 5’-GACAGCAAATGGGTCGGATCCATATGGATTTCGGAGCTTTACCC-3’ (SEQ ID NO: 3);
[0037] Reverse primer: 5’-CTCGAGTGCGGCCGCAAGCTTTCACACGACGACCTGTTTGGG-3’ (SEQ ID NO:4).
[0038] Then, using the Mycobacterium tuberculosis H37Rv genome (purchased from Shanghai Jingnuo Biotechnology Co., Ltd.) as a template and the sequences of SEQ ID NO:3 and SEQ ID NO:4 as amplification primers, the Rv1039c gene fragment was amplified by PCR (the amplified sequence is shown in SEQ ID NO:2), and then agarose gel electrophoresis was performed.
[0039] The product was recovered by electrophoresis, and the Rv1039c product was ligated in one step to the linearized pET28a plasmid (purchased from Novagen) digested with BamH I and Hind III, and then transformed into DH5α Escherichia coli competent cells (purchased from TAKARA), and spread on LB solid medium with 50 μg / mL kanamycin resistance for screening. Positive clones were picked for identification, and after correct sequencing (by Shanghai Sangon Biotech Co., Ltd.), the prokaryotic expression plasmid pET28a-Rv1039c was obtained. The confirmation results showed that in the prokaryotic expression plasmid pET28a-Rv1039c, the Rv1039c gene expression sequence was as shown in SEQ ID NO:2.
[0040] Pick the positive single colonies and place them in 4 mL of LB liquid medium containing 50 μg / mL kanamycin resistance, and culture them overnight in a constant temperature shaker at 37°C. The next day, inoculate the recombinant bacteria into LB liquid medium containing 50 μg / mL kanamycin resistance at a ratio of 1:100, culture them in a constant temperature shaker at 37°C until the OD value is between 0.4 and 0.6, add IPTG to a final concentration of 0.5 mmol / L, and induce for 6 h at 150 rpm at 37°C in a constant temperature shaker. Centrifuge to collect the bacterial cells and wash them three times with sterile PBS. Lyse the bacterial cells using an ultrasonic cell disruptor in an ice-water mixture (30 W, disrupt for 3 s, interval 5 s). Subsequently, centrifuge at 10000 rpm for 10 min to collect the supernatant. Take out the supernatant after centrifugation, and resuspend the precipitate with an equal volume of sterile PBS. Purify the supernatant collected after centrifugation using a Ni-Agarose His-tag protein purification kit. First, pretreat the column by sequentially adding 7.5 mL of sterilized ultrapure water, 12.5 mL of Charge buffer, and 7.5 mL of Binding buffer to wash the purification column with His-tag, then load the sample onto the column, and finally add 25 mL of Binding buffer, 15 mL of Washing buffer, and 15 mL of Elute buffer, cover the stopper, and let it stand at room temperature for 20 min, and collect the purified protein with a sterile 1.5 mL Eppendorf tube. In addition, perform sequencing confirmation. The amino acid sequence of the expressed protein is correct, as specifically shown in SEQ ID NO:1.
[0041] (2) The Rv1039c protein inhibits the secretion level of the T cell activation cytokine IL-2
[0042] Seed dendritic cells derived from the bone marrow of C57BL / 6 mice (BMDCs) in a 24-well cell culture plate, add 50 μg / mL of Rv1039c protein (endotoxin-free) for stimulation and culture, and set up an unstimulated control group at the same time. After 2 h of culture, add 50 μL of wild Mycobacterium smegmatis (MS) mc 2 155 to each well and continue the stimulation and culture for 4 h, then add Ag85A preserved in the laboratory 241-260Specific T cell hybridoma DE10 (Z Xu, C Meng, B Qiang, et al. Differential effects of Mycobacterium bovis BCG on macrophages and dendritic cells from murine spleen [J]. International Journal of Molecular Sciences, 2015, 16(10): 24127-24138.) was co-incubated with 1 mL for 24 h, and the culture supernatant was collected. The secretion level of IL-2 was detected by ELISA. The results showed that Rv1039c could significantly inhibit the secretion level of IL-2 ( Figure 1 ). Among them, MS represents the positive control group; Rv1039c + MS represents the Rv1039c treatment group; Rv1039c + Trypsin + MS represents the group treated with Rv1039c after trypsin digestion; Medium represents the negative control group, in which only BMDCs were co-incubated with DE10.
[0043] Example 2 Overexpression of Rv1039c inhibits the secretion of T cell activation cytokine IL-2 induced by Mycobacterium smegmatis
[0044] (1) Construction and identification of recombinant Mycobacterium smegmatis rMS::pMV261-Rv1039c
[0045] First, primers with BamH I and EcoR I double digestion sites were designed:
[0046] Forward primer: 5’-GGCCAAGACAATTGCGGATCCATGGATTTCGGAGCTTTACCC-3’ (SEQ ID NO:5)
[0047] Reverse primer: 3’-ACATCGATAAGCTTCGAATTCTCAGTGATGATGGTGATGATGCACGACGACCTGTTTGGG-5’ (SEQ ID NO:6)
[0048] Then, using the Mycobacterium tuberculosis H37Rv genome as a template and the sequences of SEQ ID NO:5 and SEQ ID NO:6 as amplification primers, the Rv1039c gene (the sequence is shown in SEQ ID NO:2) was amplified by PCR. After agarose gel electrophoresis and recovery, the purified and recovered Rv1039c amplification product was cloned into the linearized pMV261 vector obtained by BamH I and EcoR I digestion of the pMV261 vector donated by Southwest University, and then transformed into DH5α competent bacteria. Single colonies were picked, and the target fragment was amplified using inner and outer primers for identification. The positive clones were sent to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for sequencing. The correctly sequenced DH5α (pMV261-Rv1039c) was cultured on a large scale, and single colonies were picked to obtain the recombinant plasmid pMV261-Rv1039c. Subsequently, the recombinant plasmid was electrotransformed into Mycobacterium smegmatis mc 2 155 competent cells. Single colonies grown after electrotransformation were picked for PCR identification, and the successfully identified strains were named rMS::pMV261-Rv1039c.
[0049] (2) Overexpression of Rv1039c in recombinant Mycobacterium smegmatis inhibits the secretion of the T cell activation cytokine IL-2
[0050] C57BL / 6 mouse peritoneal macrophages were seeded in 24-well cell culture plates. The recombinant bacterium rMS::pMV261-Rv1039c was added to infect the cells at an MOI of 10. The rMS::pMV261 empty vector bacterium was used as a control group. After 2 h of infection, 1640 medium containing 10 μg / mL OVA 323-339 polypeptide (synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) was added, and the cells were cultured at 37 °C for 3 h. Subsequently, OVA 323-339 -specific CD4 + T cells were co-cultured with the infected peritoneal macrophages. After 24 h, the supernatant was collected, and the secretion level of IL-2 was detected by ELISA. The results showed that compared with the rMS::pMV261 empty vector bacterium, the secretion level of IL-2 in the recombinant bacterium rMS::pMV261-Rv1039c group was significantly downregulated ( Figure 2 ). Among them, Medium represents the negative control group, in which only peritoneal macrophages were co-incubated with OVA 323-339 -specific CD4 + T cells; OVA 323-339 +Medium represents the positive control group; BSA+Medium represents the isotype control group.
[0051] Example 3 Overexpression of Rv1039c inhibits the transcription of antigen-presenting cell surface molecules induced by Mycobacterium smegmatis
[0052] C57BL / 6 mouse BMDCs were seeded in 24-well cell culture plates. Recombinant bacteria rMS::pMV261-Rv1039c were added to infect the cells at a multiplicity of infection (MOI) of 10. rMS::pMV261 empty vector bacteria were used as the control group. Cells were collected at 6 h, 24 h, and 48 h after infection to extract RNA. Transcription levels of cell surface molecules such as CD40, CD80, MHC II, and CIITA were measured by fluorescence quantitative PCR, with GAPDH as the internal reference. The primers used are shown in Table 1, and the amplification system and amplification program are shown in Tables 2 and 3. The results showed that in BMDCs, compared with the rMS::pMV261 empty vector bacteria, the transcription levels of CD40, CD80, MHC II, and CIITA in the recombinant bacteria rMS:pMV261-Rv1039c group were significantly down-regulated ( Figure 3 ).
[0053] Meanwhile, the transcription levels of surface molecules CD40, CD80, MHC II, and CIITA on RAW264.7 after infection with recombinant bacteria and empty vector bacteria were detected (the steps were the same as above, replacing BMDCs with RAW264.7). The results showed that in mouse RAW264.7, compared with the rMS::pMV261 empty vector bacteria, the transcription levels of CD40, CD80, MHC II, and CIITA in the recombinant bacteria rMS::pMV261-Rv1039c group were significantly down-regulated ( Figure 4 ).
[0054] Table 1 Fluorescence quantitative PCR primers
[0055]
[0056] Table 2 Fluorescence quantitative PCR amplification system
[0057]
[0058] Table 3 Fluorescence quantitative PCR amplification program
[0059]
[0060] Example 4 Overexpression of Rv1039c inhibits the expression of MHC II on the surface of antigen-presenting cells induced by Mycobacterium smegmatis
[0061] C57BL / 6 mice BMDCs were cultured and infected with the recombinant bacterium rMS::pMV261-Rv1039c at an MOI of 10. The rMS::pMV261 empty vector bacterium was used as the control group. Cell lysates were collected at 0 h, 3 h, and 6 h after infection, and the protein expression level of MHC II was determined by Western blot. The results showed that in BMDCs, compared with the rMS::pMV261 empty vector bacterium, the expression level of MHC II in the recombinant bacterium rMS::pMV261-Rv1039c group was significantly downregulated ( Figure 5 ). Among them, Medium represents the medium control group.
[0062] Meanwhile, the protein expression levels of MHC II at 0 h, 3 h, and 6 h after the recombinant bacterium and the empty vector bacterium infected RAW264.7 cells were detected (the steps were the same as above, replacing BMDCs with RAW264.7). The results showed that in RAW264.7, compared with the rMS::pMV261 empty vector bacterium, the expression level of MHC II in the recombinant bacterium rMS::pMV261-Rv1039c group was significantly downregulated at 3 h and 6 h ( Figure 6 ). Among them, Medium represents the medium control group.
Claims
1. Use of Rv1039c protein in the preparation of an antigen presentation inhibitor, characterized in that: The amino acid sequence of the Rv1039c protein is shown in SEQ ID NO:
1.
2. Use of the gene encoding the Rv1039c protein according to claim 1 in the preparation of an antigen presentation inhibitor.
3. The use according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:
2.
4. Use of a recombinant vector, recombinant bacteria or recombinant cell containing a gene encoding Rv1039c protein in the preparation of an antigen presentation inhibitor, characterized in that: The amino acid sequence of the Rv1039c protein is shown in SEQ ID NO:
1.
5. The use according to claim 4, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:
2.
6. The use according to any one of claims 1 to 5, characterized in that: Inhibition of antigen presentation includes inhibiting the secretion of T cell activation cytokines and downregulating the transcription and protein expression levels of molecules on the surface of dendritic cells and macrophages.
7. The use according to claim 6, characterized in that: The T cell activating cytokine is IL-2.
8. The use according to claim 6, characterized in that: The surface molecules are CD40, CD80, MHC II and CIITA.
9. Use of a recombinant vector, recombinant bacteria or recombinant cell containing a gene encoding Rv1039c protein in the preparation of a drug for treating mycobacterial infection, characterized in that: The amino acid sequence of the Rv1039c protein is shown in SEQ ID NO:
1.
10. The use according to claim 9, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:2.